EGF inhibits survivin protein degradation by blocking survivin ubiquitination. (A) INS-1 cells were starved overnight then treated with or without EGF for serial time points, as indicated. Survivin mRNA levels in the presence of EGF were compared to those in the absence of EGF, as performed by quantitative RT-PCR (qRT-PCR). The relative levels and standard error (SE) of triplicate experiments are shown. (B) Primary mouse islets were starved overnight then treated with or without EGF for 2 hours. qRT-PCR was performed for all the mouse survivin splice forms (survivin 121, 140, and 40). Bars represent the relative levels of duplicate experiments. (C) MIN6 cells were transfected with a survivin promoter construct regulating luciferase (luc-6000) or control plasmid (EV). Reporter activity was measured after overnight serum-deprivation and 2 hour EGF treatment. The mean and SE of triplicate experiments is shown. (D) INS-1 cells were starved overnight then treated with EGF for 4 hours. CTX (100 mg/ml) was added at the indicated times prior to the completion of the EGF treatment and protein harvested for Western blot analysis. (E) INS-1 cells were starved overnight then treated with the proteasome inhibitor MG132 for 3 hours followed by EGF for 1 hour. Lysates were harvested and immunoprecipitated with anti-survivin or immunoglobulin (Ig) control, resolved by SDS-PAGE, transferred to PVDF membranes and immunoblotted with anti-survivin or anti-ubiquitin (pan-Ub).